Characteristics, clinical course and outcome of ventilated patients at a non-surgical intensive care unit in Germany: a single-centre, retrospective observational cohort analysis.

OBJECTIVES: The objective of this study was to evaluate epidemiological characteristics, clinical course and outcome of mechanically ventilated non-surgical intensive care unit (ICU) patients, with the aim of improving the strategic planning of ICU capacities. DESIGN: We conducted a retrospective observational cohort analysis. Data from mechanically ventilated intensive care patients were obtained by investigating electronic health records. The association between clinical parameters and ordinal scale data of clinical course was evaluated using Spearman correlation and Mann-Whitney U test. Relations between clinical parameters and in-hospital mortality rates were examined using binary logistic regression analysis. SETTING: A single-centre study at the non-surgical ICU of the University Hospital of Frankfurt, Germany (tertiary care-level centre). PARTICIPANTS: All cases of critically ill adult patients in need of mechanical ventilation during the years 2013-2015 were included. In total, 932 cases were analysed. RESULTS: From a total of 932 cases, 260 patients (27.9%) were transferred from peripheral ward, 224 patients (24.1%) were hospitalised via emergency rescue services, 211 patients (22.7%) were admitted via emergency room and 236 patients (25.3%) via various transfers. In 266 cases (28.5%), respiratory failure was the reason for ICU admission. The length of stay was higher in non-geriatric patients, patients with immunosuppression and haemato-oncological disease or those in need of renal replacement therapy. 431 patients died, which corresponds to an all-cause in-hospital mortality rate of 46.2%. 92 of 172 patients with presence of immunosuppression (53.5%), 111 of 186 patients (59.7%) with pre-existing haemato-oncological disease, 27 of 36 patients (75.0%) under extracorporeal membrane oxygenation (ECMO) therapy, and 182 of 246 patients (74.0%) undergoing renal replacement therapy died. In logistic regression analysis, these subgroups and older age were significantly associated with higher mortality rates. CONCLUSIONS: Respiratory failure was the main reason for ventilatory support at this non-surgical ICU. Immunosuppression, haemato-oncological diseases, the need for ECMO or renal replacement therapy and older age were associated with higher mortality.

Implementation and use of mHealth home telemonitoring in adults with acute COVID-19 infection: a scoping review protocol.

INTRODUCTION: mHealth refers to digital technologies that, via smartphones, mobile apps and specialised digital sensors, yield real-time assessments of patient's health status. In the context of the COVID-19 pandemic, these technologies enable remote patient monitoring, with the benefit of timely recognition of disease progression to convalescence, deterioration or postacute sequelae. This should enable appropriate medical interventions and facilitate recovery. Various barriers, both at patient and technology levels, have been reported, hindering implementation and use of mHealth telemonitoring. As systematised and synthesised evidence in this area is lacking, we developed this protocol for a scoping review on mHealth home telemonitoring of acute COVID-19. METHODS AND ANALYSIS: We compiled a search strategy following the PICO (Population, Intervention, Comparator, Outcome) and PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses recommendation for Scoping Reviews) guidelines. MEDLINE, Embase and Web of Science will be searched from 1 March 2020 to 31 August 2021. Following the title and abstract screening, we will identify, systematise and synthesise the available knowledge. Based on pilot searches, we preview three themes for descriptive evidence synthesis. The first theme relates to implementation and use of mHealth telemonitoring, including reported barriers. The second theme covers the interactions of the telemonitoring team within and between different levels of the healthcare system. The third theme addresses how this telemonitoring warrants the continuity of care, also during disease transition into deterioration or postacute sequelae. ETHICS AND DISSEMINATION: The studied evidence is in the public domain, therefore, no specific ethics approval is required. Evidence dissemination will be via peer-reviewed publications, conference presentations and reports to the policy makers.

Interobserver agreement in interpretation of chest radiographs for pediatric community acquired pneumonia: Findings of the pedCAPNETZ-cohort.

Although chest radiograph (CXR) is commonly used in diagnosing pediatric community acquired pneumonia (pCAP), limited data on interobserver agreement among radiologists exist. PedCAPNETZ is a prospective, observational, and multicenter study on pCAP. N = 233 CXR from patients with clinical diagnosis of pCAP were retrieved and n = 12 CXR without pathological findings were added. All CXR were interpreted by a radiologist at the site of recruitment and by two external, blinded pediatric radiologists. To evaluate interobserver agreement, the reporting of presence or absence of pCAP in CXR was analyzed, and prevalence and bias-adjusted kappa (PABAK) statistical testing was applied. Overall, n = 190 (82%) of CXR were confirmed as pCAP by two external pediatric radiologists. Compared with patients with pCAP negative CXR, patients with CXR-confirmed pCAP displayed higher C-reactive protein levels and a longer duration of symptoms before enrollment (p < .007). Further parameters, that is, age, respiratory rate, and oxygen saturation showed no significant difference. The interobserver agreement between the onsite radiologists and each of the two independent pediatric radiologists for the presence of pCAP was poor to fair (69%; PABAK = 0.39% and 76%; PABAK = 0.53, respectively). The concordance between the external radiologists was fair (81%; PABAK = 0.62). With regard to typical CXR findings for pCAP, chance corrected interrater agreement was highest for pleural effusions, infiltrates, and consolidations and lowest for interstitial patterns and peribronchial thickening. Our data show a poor interobserver agreement in the CXR-based diagnosis of pCAP and emphasized the need for harmonized interpretation standards.

Predictors for long-term mortality in COPD patients requiring non-invasive positive pressure ventilation for the treatment of acute respiratory failure.

INTRODUCTION: The effectiveness of non-invasive mechanical ventilation (NIV) in the management of COPD patients suffering from acute respiratory failure (ARF) as a consequence of exacerbation of the disease, is well established. However, data on long-term outcomes and their predictors, including the individual response to NIV, are scarce. OBJECTIVES: To investigate predictors for short- and long-term mortality in this study population. METHODS: A retrospective cohort study was performed including all patients admitted to the Medium Respiratory Care Unit of Maastricht University Medical Center in Maastricht, the Netherlands, with hospitalized exacerbation of COPD (H-ECOPD) with ARF requiring NIV for the first time between January 2009 and December 2011. An extensive number of potential predictors of outcomes, including the response to NIV, were determined on admission and during hospitalization. Univariate and multivariate logistic regression was used for statistical analysis. RESULTS: Seventy-eight consecutive patients with moderate to severe COPD (mean age 71.0 ± 10.7 years; 48.7% males) were included; In-hospital, 1-year and 2-year mortality rates were 14.1%, 43.6% and 56.4%, respectively. Independent risk factors for 2-year mortality were: advanced age (odds ratio(OR) 1.025; confidence interval (CI) 1.002-1.049; P = 0.037), prolonged NIV use more than 8 days (OR:1.054;CI:1.006-1.104; P = 0.027) and no successful response to NIV (OR:2.392;CI:1.297-4.413; P = 0.005). CONCLUSION: Patients with an H-ECOPD requiring NIV for the first time, constitute a severely ill patient group with high in-hospital and 2-year mortality. This study identified advanced age, NIV use more than 8 days and unsuccessful response to NIV as clinical important independent predictors for long-term mortality.

Learning in the workplace: Use of informal feedback cues in doctor-patient communication.

OBJECTIVES: We expect physicians to be lifelong learners. Participation in clinical practice is an important potential source of that learning. To support physicians in this process, a better understanding of how they learn in clinical practice is necessary. This study investigates how physicians recognise and use informal feedback from interactions with patients in outpatient settings as learning cues to adjust their communication behaviours in daily practice. METHODS: To understand physicians' use of informal feedback, we combined non-participant observations with semi-structured interviews. We enrolled 10 respiratory physicians and observed 100 physician-patient interactions at two teaching hospitals in the Netherlands. Data collection and analysis were performed iteratively according to the principles of constructivist grounded theory. RESULTS: Following stages of open, axial and selective coding, we were able to conceptualise how physicians use cues to reflect on and adjust their communication. In addition to vast variations within and across patient encounters, we observed recurring adjustments in physicians' communication behaviours in response to specific informal feedback cues. Physicians recognised and used these cues to self-monitor communication performance. They had established 'communication repertoires' based on multiple patient interactions, which many saw as learning opportunities contributing to the development of expertise. Our findings, however, show differences in physicians' individual levels of sensitivity in recognising and using learning opportunities in daily practice, which were further influenced by contextual, personal and interpersonal factors. Whereas some described themselves as having little inclination to change, others used critical incidents to fine-tune their communication repertoires, and yet others constantly reshaped them, seeking learning opportunities in their daily work. CONCLUSIONS: There is large variation in how physicians use learning cues from daily practice. To enhance learning in and from daily practice, we propose turning workplace learning into a collaborative effort with the aim of increasing awareness and the use of informal performance-relevant feedback.

Perceptions of people with respiratory problems on physician performance evaluation-A qualitative study.

BACKGROUND: Despite increasing calls for patient and public involvement in health-care quality improvement, the question of how patient evaluations can contribute to physician learning and performance assessment has received scant attention. OBJECTIVE: The objective of this study was to explore, amid calls for patient involvement in quality assurance, patients' perspectives on their role in the evaluation of physician performance and to support physicians' learning and decision making on professional competence. DESIGN: A qualitative study based on semi-structured interviews. SETTING AND PARTICIPANTS: The study took place in a secondary care setting in the Netherlands. The authors selected 25 patients from two Dutch hospitals and through the Dutch Lung Foundation, using purposive sampling. METHODS: Data were analysed according to the principles of template analysis, based on an a priori coding framework developed from the literature about patient empowerment, feedback and performance assessment. RESULTS: The analysis unearthed three predominant patient perspectives: the proactive perspective, the restrained perspective and the outsider perspective. These perspectives differed in terms of perceived power dynamics within the doctor-patient relationship, patients' perceived ability, and willingness to provide feedback and evaluate their physician's performance. Patients' perspectives thus affected the role patients envisaged for themselves in evaluating physician performance. DISCUSSION AND CONCLUSION: Although not all patients are equally suitable or willing to be involved, patients can play a role in evaluating physician performance and continuing training through formative approaches. To involve patients successfully, it is imperative to distinguish between different patient perspectives and empower patients by ensuring a safe environment for feedback.

Cell Type- and Exposure-Specific Modulation of CD63/CD81-Positive and Tissue Factor-Positive Extracellular Vesicle Release in response to Respiratory Toxicants.

Chronic exposure to respiratory stressors increases the risk for pulmonary and cardiovascular diseases. Previously, we have shown that cigarette smoke extract (CSE) triggers the release of CD63+CD81+ and tissue factor (TF)+ procoagulant extracellular vesicles (EVs) by bronchial epithelial cells via depletion of cell surface thiols. Here, we hypothesized that this represents a universal response for different pulmonary cell types and respiratory exposures. Using bead-based flow cytometry, we found that bronchial epithelial cells and pulmonary fibroblasts, but not pulmonary microvascular endothelial cells or macrophages, release CD63+CD81+ and TF+ EVs in response to CSE. Cell surface thiols decreased in all cell types upon CSE exposure, whereas depletion of cell surface thiols using bacitracin only triggered EV release by epithelial cells and fibroblasts. The thiol-antioxidant NAC prevented the EV induction by CSE in epithelial cells and fibroblasts. Exposure of epithelial cells to occupational silica nanoparticles and particulate matter (PM) from outdoor air pollution also enhanced EV release. Cell surface thiols were mildly decreased and NAC partly prevented the EV induction for PM10, but not for silica and PM2.5. Taken together, induction of procoagulant EVs is a cell type-specific response to CSE. Moreover, induction of CD63+CD81+ and TF+ EVs in bronchial epithelial cells appears to be a universal response to various respiratory stressors. TF+ EVs may serve as biomarkers of exposure and/or risk in response to respiratory exposures and may help to guide preventive treatment decisions.

Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles.

Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated t-test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers.

Risk stratification for short-term mortality at hospital admission for acute exacerbations of COPD.

BACKGROUND AND OBJECTIVE: Exacerbations of chronic obstructive pulmonary disease (ECOPD) are associated with increased in-hospital and short-term mortality. Developing an easy-to-use model to predict adverse outcomes will be useful in daily clinical practice and will facilitate management decisions. We aimed to assess mortality rates and potential predictors for short-term mortality after severe ECOPD. Classification and Regression Tree (CART) model was used to identify predictors of adverse outcome. METHODS: A retrospective observational cohort study, including all patients admitted to Maastricht University Medical Center with ECOPD between June 2011 and December 2014 was performed. The last admission was taken into account, and its demographic, clinical and biochemical data were recorded. RESULTS: A total of 364 hospitalized patients were enrolled. Mean (SD) age was 70.5 (10.2) years, 54.4% were male and mean FEV1 45.2% (17.7) of predicted. The in-hospital and 90-day mortality were, respectively, 8.5 and 16.2%. Independent risk factors for 90-day mortality were: PaC02 (odds ratio (OR): 1.31; 95% confidence interval (CI): 1.00-0.35), age (OR: 1.09; CI: 0.06-0.11), body mass index (BMI) < 18.5 kg/m2 (OR: 2.72; 95% CI: 0.53-1.47) and previous admission for ECOPD in last 2 years (OR: 1.29; 95% CI: -0.14, -0.65). The CART model selected PaCO2 ≥ 9.1 kPa, age > 80 years, BMI < 18.5 kg/m2 and previous admission for ECOPD as the most discriminatory factors. CONCLUSION: According CART analysis, high PaCO2 and age, low BMI and previous admission for ECOPD in last 2 years were the strongest predictors of 90-day mortality in patients with severe ECOPD. In absence of any of these factors, no patients died, suggesting that this model indeed enables risk stratification.

Serum levels of hyaluronic acid are associated with COPD severity and predict survival.

Hyaluronic acid (HA) and its degradation products play an important role in lung pathophysiology and airway remodelling in chronic obstructive pulmonary disease (COPD).We investigated if HA and its degrading enzyme hyaluronidase (HYAL)-1 are associated with COPD severity and outcome.Serum HA was assessed in a discovery cohort of 80 COPD patients at stable state and exacerbations. HA, HYAL-1 and HYAL-1 enzymatic activity were evaluated at stable state, exacerbations and 4 weeks after exacerbations in 638 COPD patients from the PROMISE validation cohort.In the discovery cohort, serum HA was higher at exacerbations compared with the stable state (p=0.015). In the validation cohort, HA was higher at moderate and severe exacerbations than at baseline (p<0.001), and remained higher after 4 weeks (p<0.001). HA was strongly predictive for overall survival since it was associated with time to death (p<0.001) independently of adjusted Charlson score, annual exacerbation rate and BODE (body mass, airflow obstruction, dyspnoea, exercise capacity) index. Serum HYAL-1 was increased at moderate (p=0.004) and severe (p=0.003) exacerbations, but decreased after 4 weeks (p<0.001). HYAL-1 enzymatic activity at stable state was inversely correlated with FEV1 % pred (p=0.034) and survival time (p=0.017).Serum HA is associated with COPD severity and predicts overall survival. Degradation of HA is associated with airflow limitation and impairment of lung function.

Box-ticking and Olympic high jumping - Physicians' perceptions and acceptance of national physician validation systems.

PURPOSE: National physician validation systems aim to ensure lifelong learning through periodic appraisals of physicians' competence. Their effectiveness is determined by physicians' acceptance of and commitment to the system. This study, therefore, sought to explore physicians' perceptions and self-reported acceptance of validation across three different physician validation systems in Europe. MATERIALS AND METHODS: Using a constructivist grounded-theory approach, we conducted semi-structured interviews with 32 respiratory specialists from three countries with markedly different validation systems: Germany, which has a mandatory, credit-based system oriented to continuing professional development; Denmark, with mandatory annual dialogs and ensuing, non-compulsory activities; and the UK, with a mandatory, portfolio-based revalidation system. We analyzed interview data with a view to identifying factors influencing physicians' perceptions and acceptance. RESULTS: Factors that influenced acceptance were the assessment's authenticity and alignment of its requirements with clinical practice, physicians' beliefs about learning, perceived autonomy, and organizational support. CONCLUSIONS: Users' acceptance levels determine any system's effectiveness. To support lifelong learning effectively, national physician validation systems must be carefully designed and integrated into daily practice. Involving physicians in their design may render systems more authentic and improve alignment between individual ambitions and the systems' goals, thereby promoting acceptance.

Extracellular vesicles released in response to respiratory exposures: implications for chronic disease.

Extracellular vesicles (EV) are secreted signaling entities that enhance various pathological processes when released in response to cellular stresses. Respiratory exposures such as cigarette smoke and air pollution exert cellular stresses and are associated with an increased risk of several chronic diseases. The aim of this review was to examine the evidence that modifications in EV contribute to respiratory exposure-associated diseases. Publications were searched using PubMed and Google Scholar with the search terms (cigarette smoke OR tobacco smoke OR air pollution OR particulate matter) AND (extracellular vesicles OR exosomes OR microvesicles OR microparticles OR ectosomes). All original research articles were included and reviewed. Fifty articles were identified, most of which investigated the effect of respiratory exposures on EV release in vitro (25) and/or on circulating EV in human plasma (24). The majority of studies based their main observations on the relatively insensitive scatter-based flow cytometry of EV (29). EV induced by respiratory exposures were found to modulate inflammation (19), thrombosis (13), endothelial dysfunction (11), tissue remodeling (6), and angiogenesis (3). By influencing these processes, EV may play a key role in the development of cardiovascular diseases and chronic obstructive pulmonary disease and possibly lung cancer and allergic asthma. The current findings warrant additional research with improved methodologies to evaluate the contribution of respiratory exposure-induced EV to disease etiology, as well as their potential as biomarkers of exposure or risk and as novel targets for preventive or therapeutic strategies.

Doctors on the move: a European case study on the key characteristics of national recertification systems.

OBJECTIVES: With increased cross-border movement, ensuring safe and high-quality healthcare has gained primacy. The purpose of recertification is to ensure quality of care through periodically attesting doctors' professional proficiency in their field. Professional migration and facilitated cross-border recognition of qualifications, however, make us question the fitness of national policies for safeguarding patient care and the international accountability of doctors. DESIGN AND SETTING: We performed document analyses and conducted 19 semistructured interviews to identify and describe key characteristics and effective components of 10 different European recertification systems, each representing one case (collective case study). We subsequently compared these systems to explore similarities and differences in terms of assessment criteria used to determine process quality. RESULTS: Great variety existed between countries in terms and assessment formats used, targeting cognition, competence and performance (Miller's assessment pyramid). Recertification procedures and requirements also varied significantly, ranging from voluntary participation in professional development modules to the mandatory collection of multiple performance data in a competency-based portfolio. Knowledge assessment was fundamental to recertification in most countries. Another difference concerned the stakeholders involved in the recertification process: while some systems exclusively relied on doctors' self-assessment, others involved multiple stakeholders but rarely included patients in assessment of doctors' professional competence. Differences between systems partly reflected different goals and primary purposes of recertification. CONCLUSION: Recertification systems differ substantially internationally with regard to the criteria they apply to assess doctors' competence, their aims, requirements, assessment formats and patient involvement. In the light of professional mobility and associated demands for accountability, we recommend that competence assessment includes patients' perspectives, and recertification practices be shared internationally to enhance transparency. This can help facilitate cross-border movement, while guaranteeing high-quality patient care.

Redox-dependent thiol modifications: implications for the release of extracellular vesicles.

Extracellular vesicles (EVs), including microvesicles and exosomes, are emerging as important regulators of homeostasis and pathophysiology. During pro-inflammatory and pro-oxidant conditions, EV release is induced. As EVs released under such conditions often exert pro-inflammatory and procoagulant effects, they may actively promote the pathogenesis of chronic diseases. There is evidence that thiol group-containing antioxidants can prevent EV induction by pro-inflammatory and oxidative stimuli, likely by protecting protein thiols of the EV-secreting cells from oxidation. As the redox state of protein thiols greatly impacts three-dimensional protein structure and, consequently, function, redox modifications of protein thiols may directly modulate EV release in response to changes in the cell's redox environment. In this review article, we discuss targets of redox-dependent thiol modifications that are known or expected to be involved in the regulation of EV release, namely redox-sensitive calcium channels, N-ethylmaleimide sensitive factor, protein disulfide isomerase, phospholipid flippases, actin filaments, calpains and cell surface-exposed thiols. Thiol protection is proposed as a strategy for preventing detrimental changes in EV signaling in response to inflammation and oxidative stress. Identification of the thiol-containing proteins that modulate EV release in pro-oxidant environments could provide a rationale for broad application of thiol group-containing antioxidants in chronic inflammatory diseases.

IFNΛ3/4 locus polymorphisms and IFNΛ3 circulating levels are associated with COPD severity and outcomes.

BACKGROUND: Interferon lambdas (IFNLs) have important anti-viral/bacterial and immunomodulatory functions in the respiratory tract. How do IFNLs impact COPD and its exacerbations? METHODS: Five hundred twenty eight patients were recruited in a prospective observational multicentre cohort (PROMISE) study. The genetic polymorphisms (rs8099917 and rs12979860) within the IFNL3/4 gene region and circulating levels of IFNL3 in COPD patients were determined and associated with disease activity and outcome during a median follow-up of 24 months. RESULTS: The GG genotype significantly influenced severe exacerbation rate (42 vs. 23%; p = 0.032) and time to severe exacerbation (HR = 2.260; p = 0.012). Compared to the TT or TG genotypes, the GG genotype was associated with severe dyspnoea (modified medical research council score ≥ median 3; 22 vs 42%, p = 0.030). The CC genotype of the rs12979860 SNP was associated with a poorer prognosis (body mass index, airflow obstruction, dyspnea and exercise capacity index ≥ median 4; 46 vs. 36% TC vs. 20.5% TT; p = 0.031). Patients with stable COPD and at exacerbation had significantly lower circulating IFNL3 compared to healthy controls (p < 0.001 and p < 0.001, respectively). Circulating IFNL3 correlated to post-bronchodilator FEV1%predicted and the tissue maturation biomarker Pro-collagen 3. CONCLUSION: IFNL3/4 polymorphisms and circulating IFNL3 may be associated with disease activity and outcomes in COPD. TRIAL REGISTRATION: Clinical Trial registration http://www.isrctn.com/ identifier ISRCTN99586989 on 16 April 2008.

Increased Small Intestinal Permeability during Severe Acute Exacerbations of COPD.

BACKGROUND: Disturbances of intestinal integrity, manifested by increased gastro-intestinal (GI) permeability, have been found in chronic obstructive pulmonary disease (COPD) patients during physical activity, often associated with intermittent hypoxic periods. Evidence about extrapulmonary organ disturbances, especially of the GI tract, during hospitalised acute exacerbation of COPD (AE-COPD) with hypoxaemic respiratory failure (RF) is lacking. OBJECTIVE: The aim was to assess changes in GI permeability in patients with AE-COPD and during recovery 4 weeks later. METHODS: All patients admitted to our hospital with AE-COPD accompanied by hypoxaemia at admission (PaO2 <8.7 kPa or O2 saturation <93%) were screened between October 2013 and February 2014. Patients with a history of GI or renal disease, chronic heart failure, or use of non-steroidal anti-inflammatory drugs in the 48 h before the test were excluded. GI permeability was assessed by evaluating urinary excretion ratios of the orally ingested sugars lactulose/L-rhamnose (L/R ratio), sucrose/L-rhamnose (Su/R ratio) and sucralose/erythritol (S/E ratio). RESULTS: Seventeen patients with severe to very severe COPD completed the study. L/R ratio (×103) at admission of AE-COPD was significantly higher than in the recovery condition (40.9 [29.4-49.6] vs. 27.3 [19.5-47.7], p = 0.039), indicating increased small intestinal permeability. There were no significant differences in the individual sugar levels in urine nor in the 0- to 5-h urinary S/E and Su/R ratios between the 2 visits. CONCLUSION: This is the first study showing increased GI permeability during hospitalised AE-COPD accompanied by hypoxaemic RF. Therefore, GI integrity in COPD patients is an attractive target for future research and for the development of interventions to alleviate the consequences of AE-COPD.

Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies.

Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules.

Cigarette smoke extract induced exosome release is mediated by depletion of exofacial thiols and can be inhibited by thiol-antioxidants.

INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) with pathological properties when exposed to cigarette smoke extract (CSE). As CSE causes oxidative stress, we investigated whether its oxidative components are responsible for inducing EV release and whether this could be prevented using the thiol antioxidants N-acetyl-l-cysteine (NAC) or glutathione (GSH). METHODS: BEAS-2B cells were exposed for 24h to CSE, H2O2, acrolein, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), bacitracin, rutin or the anti-protein disulfide isomerase (PDI) antibody clone RL90; with or without NAC or GSH. EVs in media were measured using CD63+CD81+ bead-coupled flow cytometry or tunable resistive pulse sensing (TRPS). For characterization by Western Blotting, cryo-transmission electron microscopy and TRPS, EVs were isolated using ultracentrifugation. Glutathione disulfide and GSH in cells were assessed by a GSH reductase cycling assay, and exofacial thiols using Flow cytometry. RESULTS: CSE augmented the release of the EV subtype exosomes, which could be prevented by scavenging thiol-reactive components using NAC or GSH. Among thiol-reactive CSE components, H2O2 had no effect on exosome release, whereas acrolein imitated the NAC-reversible exosome induction. The exosome induction by CSE and acrolein was paralleled by depletion of cell surface thiols. Membrane impermeable thiol blocking agents, but not specific inhibitors of the exofacially located thiol-dependent enzyme PDI, stimulated exosome release. SUMMARY/CONCLUSION: Thiol-reactive compounds like acrolein account for CSE-induced exosome release by reacting with cell surface thiols. As acrolein is produced endogenously during inflammation, it may influence exosome release not only in smokers, but also in ex-smokers with chronic obstructive pulmonary disease. NAC and GSH prevent acrolein- and CSE-induced exosome release, which may contribute to the clinical benefits of NAC treatment.